Experiments on Sterilization Methods: Autoclaving, Filtration, and UV Light in Bacterial Control Report

Introduction

Sterilization is integral to all medical actions requiring a clean environment. It is used when there is contact between the sterile tissues of the patient and the surgical instrument or medical device, which carriesthe risk of introducing various infections. Therefore, the inability of medical professionals to sterilize the equipment leads to increased risks of infection transmission, including different environmental pathogens and viruses (Rutala & Weber, 2018). Similar assumptions are applicable in the laboratory setting when studying particular bacteria.

The aseptic technique unites all sterilization types and is the general term for these practices. It describes the combination of medical and laboratory procedures that allow specialists to protect patients from microorganisms, viruses, and bacteria and to study the pure culture in the laboratory. It differs from sterilization because it involves using barriers to prevent contamination, including masks, sterile drapes, sterile gowns, and gloves (Bykowski & Stevenson, 2020).

The clean skin of the professional in the laboratory or the medical setting is a critical issue because human skin is where many microorganisms and bacteria are present (Bykowski & Stevenson, 2020). In the laboratory setting, the contamination will ruin the objective results obtained from the experiment. Aseptic techniques are essential in medical practice because they decrease the chances of inaccurate contamination of the patient (Bykowski & Stevenson, 2020). Therefore, aseptic techniques aim to minimize the possibility of contamination, while sterile techniques eliminate pathogens.

It is possible to introduce four significant sterilization types used in laboratory practice. They include UV light, moist heat, filtration, and autoclaving. Autoclaving is a highly effective method of sterilization for items that are not heat-sensitive (General Microbiology Laboratory Manual, 2022). Autoclaving will eliminate all spores by heating each item to 121 degrees Celsius at 15 pounds per square inch for 15 minutes (General Microbiology Laboratory Manual, 2022).

Bacteria are killed via moist heat sterilization, which involves placing them in a boiling water bath. Filtration sterilizes heat-sensitive liquids by removing microorganisms from liquid culture. UV ray exposure can also render bacteria inert. By producing thymine dimers, which lead to mutations, radiation exposure harms bacterial DNA. Each method is suitable for a particular variety of bacteria. After each technique is finished, the bacteria will be checked, and the degree of sterilization will be compared.

Materials and Methods

The first technique involves autoclaving and moist heat. The procedure consists of the subsequent steps that allow reaching the objective results using these methods:

1. Get four tubes. Spore strips, also called Bacillus stearothermophilus, are seen in 3 tubes.
2. The control tube will not be heated; it will be one of the tubes with a strip. The fourth sterile tube, which does not have a spore strip, will be quality control for the aseptic pipetting method.
3. Reserve one tube so it can be autoclaved. 15 minutes in an autoclave
4. The second tube must spend 15 minutes in the hot water bath.
5. Fill the four tubes with two ccs of tryptic soy broth after the heating treatments.
6. Keep the incubator at 56°C until the following lab time.
7. The findings are described as growth (+/-) or absence (+/-) (McCormick et al., 2018).

The required materials for this experiment are a 56 degrees Celsius incubator, tryptic soy broth, ahot plate, aboiling water bath, test tubes, apipette, and Bacillus stearothermophilus spore strips.

The second method used for the experiment is filtration, and it supposes the use of the following materials:

• 37 degrees Celsius incubator;
• Non-sterile glucose (5%) (msg);
• Pipette;
• Tryptic soy broth (TSB);
• Sterile test tubes;
• Syringe;
• Filter (2mm width).

The method of filtration includes the following steps:

1. Get four sterile test tubes.
2. Create three tubes with 2 mL of sterile tryptic soy broth.
3. Fill one tube with TSB and add 100 mL (0.1 mL) of non-sterile glucose solution (5%) to the tube.
4. Put the final sterile tube of filter-sterilized glucose into the process (tube without TSB).
5. Fill another medium tube with 100 mL (0.1 mL) of this pure glucose.
6. Until the next lab session, incubate at 37°C (the third tube is a control and only contains TSB).
7. Record the findings as growth (+/-) or absence (+/-) (McCormick et al., 2018).

The third method used in this laboratory report is the effect of UV light. The method consists of the following steps:

1. Use a sterile swab to cover the whole surface of a Tryptic Soy Agar plate with a bacterial culture (make sure to mix the culture first to resuspend all cells).
2. Separate the plate into six portions and identify each with the following times: 0, 0.5, 1, 3, 5, and 7. (As seen in the figure.)
3. At the UV light station, remove the lid from the petri dish and cover all but the 7-minute portion of the plate with a 4×6″ card before exposing the plate to UV light. Move the card down once 2 minutes are up so that the 5-minute area is visible. Move the card to reveal the 3 0 min0.5 min1 min7 min3 min5 min area after another 2 minutes. Repeat to reveal the one-minute portion after an additional two minutes. Move the card so that the portion with 0 minutes of exposure is still covered after 30 seconds, then expose it for 30 seconds.
4. While repeating with other organisms, cover the plates to protect them from light.
5. Place the plates in the drawers on the bench and completely wrap the two plates in aluminum foil. Incubate at room temperature until the end of the following lab period.
6. Take note of any growth, absence of growth, or relative quantity of growth in comparison to organisms that were not exposed. Note any variations in the bacteria’s appearance as well.

It is necessary to use the following materials during the experiment with UV light:

• Swabs;
• Broth culture of Serratia marcescens;
• 4”x6” card;
• UV light station;
• Aluminum foil;
• Tryptic soy agar (TSA) plate;
• Micrococcus luteus.

Results

The results for autoclaving and moist heat are represented in Table 1:
(+ = bacterial growth present, – = growth absent)

Table 1. Results for Autoclaving and Moist Heat

The results of filtration are represented in Table 2:

Table 2. Results of Filtration

It is possible to illustrate the results of exposing to UV light with Table 3:

Table 3. The Results of Disinfection by Exposure to UV Light

Discussion

Autoclaving

The autoclaving and moist heat results show that all observed outcomes aligned with expectations. Since there was nothing to impede bacterial growth, growth was anticipated with the control strip. Because no Bacillus stearothermophilus spores were present, the control without the strip displayed no growth. The absence of growth in the spore-free control meant the tubes were uncontaminated. Since autoclaving is a very efficient aseptic procedure, little bacterial growth was anticipated; the findings supported this.

Moist Heat

Growth was visible in the tube submerged in hot water. Being a thermophile, Bacillus stearothermophilus has excellent heat tolerance, which explains this bacterium’s choice. Because of this, boiling failed to eradicate the microorganisms, which supports the experiment’s aim.

Filtration

According to the filtration results, the two control tube results were what was anticipated. No germs could grow because none were present in either tube. The method uses TSB and glucose to show the bacterial growth, corresponding to the experiment’s goal. There was no contamination because there were no microorganisms in the controls.

The results from the tubes containing TSB and glucose were inconsistent with what was predicted. Nut growth was anticipated, but the tube containing TSB and non-sterilized glucose showed no growth. Bacteria should have been created by unfiltered glucose.

While no growth was anticipated, the tube containing TSB and sterilized glucose showed growth. Bacterial growth ought to have been stopped by filtered glucose. The incorrect labeling of the TSB and glucose tubes was most likely the cause of the unexpected results. Since there was no contamination, the anticipated outcomes would have been achieved if the labels had been replaced.

Exposure to UV Light

The experiment’s results with exposure to UV light show that prolonged exposure to electromagnetic radiation with wavelengths below 300 nm kills bacteria. Although the experiment did not kill the anticipated germs, UV radiation effectively killed microorganisms. After about three minutes of exposure to UV radiation, S. marcescens began to exhibit a slowdown in growth.

This outcome, however, was different from what was anticipated. Since S. marcescens is capable of repairing its DNA, UV radiation should not have had an impact on bacterial growth. The M. luteus plate grew over the whole seven-minute UV light exposure. The UV radiation should have produced thymine dimers, which would have led to mutations that would have prevented M. luteus from repairing its DNA, halting bacterial development.

The two expected results were obtained, but on the wrong plate; therefore, these results were probably caused by the incorrect labeling of our TSA plates. S. marcescens and M. luteus show the effect of UV light, which explains the choice of these bacteria. The test shows the essence of sterilization in the laboratory setting using UV light.

Conclusion

This experiment tested various aseptic procedures to see how well they might eradicate microorganisms. The findings demonstrated that particular types of bacteria could be killed more successfully using particular aseptic approaches than others. This experiment also demonstrated the need for accurately labeling samples. Even if the experiment was conducted correctly, improper labeling could result in mistakes in the final data.

References

Bykowski, T., & Stevenson, B. (2020). Aseptic technique. Current Protocols in Microbiology, 56(1), e98. Web.

General microbiology laboratory manual. (2022). Department of Biological Sciences at Old Dominion University.

McCormick, P. J., Schoene, M. J., Pedeville, D., Kaiser, J. J., & Conyer, J. (2018). Verification testing of biological indicators for moist heat sterilization. Biomedical Instrumentation & Technology, 52(3), 199–207. Web.

Rutala, W. A., & Weber, D. J. (2018). Disinfection, sterilization, and control of hospital waste. Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases, 3294–3309.e4. Web.